The latex agglutination assay is a laboratory method to check for presence of antibodies or antigens in bodily fluids;saliva, urine, cerebrospinal fluid, or blood. The test depends on what type of sample is needed.

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Samples are sent to a lab, where it is mixed with latex beads coated with the specific antibody or antigen. If a affinity reactive substance is present, the latex beads will clump together (agglutinate).

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For example, a child has strep throat, a throat swab is taken. The sample is mixed with latex beads that are coated with antibodies against the bacteria. The bacteria in the sample will react with the antibodies on the latex particles causing clumping (agglutination).

Revision as of 14:10, 26 March 2010

If you are planning on performing an IP experiment, then consider to run a preliminary western blot first in order to get a feel for what the antibody is capable of binding.

RIPA (Radio ImmunoPreciptation Assay) buffer is a traditional name for an array of recipes that have found success over the years. Below are details that can contribute to optimizing your immunoprecipitation experiment. Lysis buffer components can and will influence the efficiency of the IP reaction. Detergent composition is a major factor for IP reactions. Adjusting salt concentration and detergent composition will influence the efficiency of the IP reaction. Membrane bound proteins (proteins based in lipid rafts), protein complexes, and protein charge can all influence the efficient yield of your gene product in the lysate prep.

The amount of protein present in cells will vary with cell type. 1 to 10 million cells can yield ~1mg of protein.

Misc. Phosphatase Inhibitors: Phosphorylation/dephosphorylation of proteins influences the charge-charge relationships that proteins have with eachother in solution. Proteins undergo covalent attachment of a phosphoryl group (phosphorylation) typically at serine, threonine, or tyrosine residues. Phosphate groups are removeable via protein phosphatases. During the extraction of phosphorylated proteins from cell and tissue, preserving the phosphorylation states of total protein is a good technique.

Protease Inhibitor Cocktails

Tableted formulations containing water-soluble protease inhibitors are user-friendly and effective substitutes in lysis buffers. When using divalent/trivalent columns for enrichment of phosphoproteins, be sure to avoid EDTA/EGTA containing cocktails.

Protein A/G/L Agarose

Protein A/G/L are common to covalently couple by cyanogen bromide to highly cross-linked 4% agarose beads. This type of matrix is stable in most aqueous buffers.

Typical Ligand density: ~3 ug Protein/ul of bead

Binding capacity: ~15-20 ug Ig/ul bead

Bead structure: 4-6% cross-linked agarose

Bead diameter: 40-170 um

Temperature stability: 4-40 C

Protein A

Native protein A is a single chain (predicted 42 kDa, SDS-PAGE 46 kDa), glycoslyation-free, cell wall component produced in strains of Staphylococcus aureus. Protein A binds specifically to the Fc region of immunoglobulin molecules, including IgG. Protein A has four high-affinity (Ka = 108/mole) binding sites toward Fc region of IgG of several species (two sites can bind at a time). Protein A is heat-stable and retains conformation even after exposure to denaturing reagents such as 4 M urea, 4 M thiocyanate and 6 M guanidine hydrochloride.

Protein L

Native protein L is an kappa light chain specific Ig-binding protein that originates from the bacteria Peptostreptococcus magnus. Protein L binds Igs through interactions with the light chains. Because no part of the heavy chain is involved in the binding interaction, Protein L binds a wider range of Ig classes than Protein A or G. Protein L binds to representatives of all classes of Ig, including IgG, IgM, IgA, IgE and IgD. Single chain variable fragments (ScFv) and Fab fragments also bind to Protein L.

In humans and mice, kappa (k) light chains predominate. The remaining immunoglobulins have lambda (l) light chains. Furthermore, Protein L is effective in binding only certain subtypes of kappa light chains. For example, it binds human VkI, VkIII and VkIV subtypes but does not bind the VkII subtype. Binding of mouse immunoglobulins is restricted to those having VkI light chains.

Protein L matrix is suitable for purification of kappa light chain-containing monoclonal antibodies from ascites or culture. Protein L is useful for purification of VLk-containing monoclonal antibodies from culture supernatant because it does not bind bovine immunoglobilins, which can be present in media. Protein L does not interfere with the antigen-binding site of the antibody.

Aprotinin activity is measured by KIU (KIU = Kallikrein Inhibitory Unit) Since the vial contains other components which makes the total dry. I recommend the following procedure to be used with this product: The normal working concentration range for aprotinin is either 0.5ug-2ug/ml (protein weight/volume) or 10 KIU-100 KIU/ml (units/volume). 1ug aprotinin/ml of RIPA buffer works well.

Lysis Buffer 2:

An SDS free lysis buffer to consider with Co-IP approaches. Used this one in graduate school to IP EGFR effectively.

Adherent cell sample preparation

Remove culture medium and rinse a subconfluent, 100 mm cell culture plate (80% confluent plate yields ~600-1000 ug protein total) with PBS at room temperature. The following steps should be performed on ice or at 4° C using fresh, ice cold buffers.

Optional: For monolayer cells, do a trypsin treatment to lift cells off the flasks prior to adding the RIPA buffer, instead of scraping the cells for a more gentle approach. If you are running a time course experiment, this is not feasible since the cells must be arrested and lysed immediately.

Gently rock plates for 15 minutes at 4° C or let the plates set on ice. This step will allow the lysis buffer to act on the cells and will increase the total yield of soluble protein. Scrape the adherent cells with a cell scraper and then transfer the scraped lysate into a sterile microcentrifuge tube. Place the tube on ice.

Optional: wash the plate once with 0.2 ml of RIPA buffer and combine with first lysate. When running multiple plates this can be tedious and not necessary if enough attention is given to the initial harvest.

Optional: Add 10 µl of 10 mg/ml PMSF stock to the lysate. If a protease inhibtior cocktail is used fresh with the RIPA buffer, this is not necessary.

Sonicate each sample on a 70% duty cycle or less by placing only the very tip of the pin into the vial, then slowly lowering it into the lysate until it foams completely and then stop. Alternatively, pass the lysate through a 21 gauge needle to shear the DNA & incubate 30–60 minutes on ice.

Microcentrifuge cell lysates at 12,000xg for 15 minutes at 4°C.

The supernatant fluid is the total cell lysate. Transfer the supernatant to a new microfuge tube and discard the pellet. Quantitate the protein amount by Bradford or BCA.

Transfer to microcentrifuge tube(s) and centrifuge at 12,000xg for 15 minutes at 4° C. The supernatant fluid is the total cell lysate. Transfer the supernatant to a new microfuge tube. This is your whole cell lysate. For increased protein recovery, resuspend the pellet in a small volume of RIPA, centrifuge and combine supernantants.

The supernatant fluid is the total cell lysate. Transfer the supernatant to a new microfuge tube and discard the pellet. Quantitate the protein amount by Bradford or BCA.

Tissue extract sample preparation

There are a few approaches to optimizing protein yield from whole tissue extract. For whole animal studies, arresting the covalent modification state of the entire proteome is essential to obtaining accurate data about the treatment or phenotype effect on the tissue being extracted.

2) Immediately heat denature the organ from the sacrificed animal by microwave in a sealed container

Weigh tissue and dice into very small pieces using a clean razor blade. Frozen tissue should be sliced very thinly and thawed in RIPA buffer containing (Protease Inhibitors) and/or (Phosphatase Inhibitors). Use 3 ml of ice cold RIPA buffer per gram of tissue.

Further disrupt and homogenize tissue with a dounce homogenizer or a sonicator, maintaining temperature at 4° C throughout all procedures. (Optional: Add 30 µl of 10 mg/ml PMSF (sc-3597) stock per gram of tissue.) Incubate on ice for 30 minutes.

Transfer to microcentrifuge tubes, centrifuge at 10,000xg for 10 minutes at 4° C. Remove supernatant and centrifuge again. The supernatant fluid is the total cell lysate. A longer centrifugation may be necessary to obtain a clear lysate.

The supernatant fluid is the total cell lysate. Transfer the supernatant to a new microfuge tube and discard the pellet. Quantitate the protein amount by Bradford or BCA.

What is Protein G PLUS? Protein G PLUS is a genetically engineered form of streptococcal Protein G that has an increased capacity and has had the albumin binding site removed to reduce background.

III) Cap tubes and incubate at 4 C on a rocker platform or rotating device for 2 hour to overnight.

IV) Collect pellet by centrifugation at 2,500 rpm (approximately 1,000xg) for 5-10 seconds. A touch spin will work. With enough samples, gravity will pellet the beads as well.

V) Carefully aspirate and discard supernatant. The trick here is to slowly aspirate with the needle touching just the top of the liquid and slowly draw down so that the needle is pulling at the surface tension of the supernatant. This will ensure no loss of beads.

Latex Agglutination Assay

The latex agglutination assay is a laboratory method to check for presence of antibodies or antigens in bodily fluids;saliva, urine, cerebrospinal fluid, or blood. The test depends on what type of sample is needed.

Samples are sent to a lab, where it is mixed with latex beads coated with the specific antibody or antigen. If a affinity reactive substance is present, the latex beads will clump together (agglutinate).

For example, a child has strep throat, a throat swab is taken. The sample is mixed with latex beads that are coated with antibodies against the bacteria. The bacteria in the sample will react with the antibodies on the latex particles causing clumping (agglutination).